MicroRNA and methods for inhibiting same

ABSTRACT

The invention relates to isolated DNA or RNA molecules comprising at least ten contiguous bases having a sequence in a pancreatic islet microRNA. In another embodiment, the invention relates to isolated single stranded pancreatic islet microRNA molecules or anti-pancreatic islet microRNA molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/745,163, filed Jan. 18, 2013, issued as U.S. Pat. No. 8,697,859 onApr. 15, 2014; which is a divisional of U.S. application Ser. No.13/306,322, filed Nov. 29, 2011, issued as U.S. Pat. No. 8,383,807 onFeb. 26, 2013; which is a divisional of U.S. application Ser. No.12/498,020, filed Jul. 6, 2009, issued as U.S. Pat. No. 8,088,914 onJan. 3, 2012; which is a divisional of U.S. application Ser. No.12/045,484, filed Mar. 10, 2008, issued as U.S. Pat. No. 7,585,969 onSep. 8, 2009; which is a divisional of U.S. application Ser. No.10/824,633 filed Apr. 13, 2004, issued as U.S. Pat. No. 7,365,058 onApr. 28, 2008, the entirety of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

MicroRNAs are typically small RNA molecules of generally about nineteento twenty-five nucleotides in length. These microRNAs are non-codingRNAs which are cleaved from hairpin precursors. Several microRNAs havebeen identified in the genomes of a wide range of multicellular lifeforms.

Many microRNAs are conserved in sequence between distantly relatedorganisms, and exhibit tissue-specific or developmental stage-specificexpression. The conservation of the sequence between organisms indicatesthat microRNAs may play important roles in biological processes.

MicroRNA molecules have been reported to control gene expression in asequence specific manner in a wide variety of organisms by blockingtranslation after partially hybridizing to the non-coding 3′ region ofmRNAs of target genes. The genes targeted by microRNAs largely remain tobe characterized.

However, there is growing evidence that microRNAs are implicated invarious diseases and illnesses. For instance, drosophilia microRNAs havebeen shown to target genes involved in apoptosis. Also, B-cell chroniclymphocytic leukemia has been linked to the deletion of two microRNAs.

Pancreatic islet cells (also referred to as islets of Langerhans) aregroups of specialized cells that make and secrete hormones. It isreported that there are five types of cells in an islet: alpha, beta,delta, PP and D1 cells.

Some of these cells are said to be involved in the regulation ofglucose. For example, alpha cells secrete glucagon which are hormonesinvolved in raising the level of glucose in the blood. Further, betacells secrete insulin, a hormone that helps the body utilize glucose forenergy.

Interference in the regulation of glucose utilization, particularly ofthe insulin-secreting beta cells, may lead to diseases and illness suchas diabetes. Therefore, it is important to elucidate the mechanismsinvolved in mediating genes which play a role in the regulation ofglucose homeostasis. For example, it is not known in the prior artwhether microRNAs, if present, mediate glucose utilization.

Thus, there is a need for materials and methods that can help elucidatethe function of regulators, such as microRNAs, of pancreatic isletcells.

Further, due to the ability of microRNAs to induce RNA degradation orrepress translation of mRNA, which encode important proteins, there isalso a need for novel molecules that inhibit pancreatic microRNA-inducedcleavage or translation repression of target mRNAs.

SUMMARY OF THE INVENTION

In one embodiment, the present invention relates to isolated DNA or RNAmolecules. The molecules comprise at least ten contiguous bases having asequence in a pancreatic islet microRNA shown in SEQ ID NOs:1-20, exceptthat up to thirty percent of the bases may be wobble bases, and up to10% of the contiguous bases may be non-complementary.

In another embodiment, the invention relates to modified single strandedpancreatic islet microRNA molecules. The molecules comprise a minimum often moieties and a maximum of fifty moieties on a molecular backbone,the molecular backbone comprising backbone units, each moiety comprisinga base bonded to a backbone unit wherein at least ten contiguous baseshave the same sequence as a contiguous sequence of bases in a pancreaticislet microRNA molecule shown in SEQ ID NOs:1-20, except that up tothirty percent of the bases pairs may be wobble base pairs, and up to10% of the contiguous bases may be additions, deletions, mismatches, orcombinations thereof; no more than fifty percent of the contiguousmoieties contain deoxyribonucleotide backbone units, and at least onemoiety is not an unmodified deoxyribonucleotide moiety or an unmodifiedribonucleotide moiety.

In a further embodiment, the invention relates to isolated singlestranded anti-pancreatic islet microRNA molecules. The moleculescomprise a minimum of ten moieties and a maximum of fifty moieties on amolecular backbone, the molecular backbone comprising backbone units,each moiety comprising a base bonded to a backbone unit, each baseforming a Watson-Crick base pair with a complementary base wherein atleast ten contiguous bases have a sequence complementary to a contiguoussequence of bases in any one of the pancreatic islet microRNA moleculesshown in SEQ ID NOs; 1-20, except that up to thirty percent of the basepairs may be wobble base pairs, and up to 10% of the contiguous basesmay be additions, deletions, mismatches, or combinations thereof; nomore than fifty percent of the contiguous moieties containdeoxyribonucleotide backbone units; and the molecule is capable ofinhibiting microRNP activity.

In yet another embodiment, the invention relates to a method forinhibiting microRNP activity in a cell. The microRNP comprises apancreatic islet microRNA molecule. The method comprises introducinginto the cell a single-stranded anti-pancreatic islet microRNA molecule,wherein the anti-pancreatic islet microRNA is complementary to thepancreatic islet microRNA molecule.

In yet a further embodiment, the invention relates to a method fortreating diabetes in a mammal in need thereof. The method comprisesintroducing into the mammal an effective amount of an anti-pancreaticislet microRNA molecule having at least ten contiguous bases having asequence shown in SEQ ID NOs:41 or 51.

In another embodiment, the invention relates to isolated microRNPscomprising an isolated DNA or RNA molecule in accordance with thepresent invention.

In yet another embodiment, the invention relates to isolate microRNPscomprising an isolated single stranded pancreatic islet microRNAmolecule in accordance with the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the modified nucleotide units discussed in thespecification. B denotes any one of the following nucleic acid bases:adenosine, cytidine, guanosine, thymine, or uridine.

FIG. 2: Predicted precursor structure and tissue expression of mousemiR-375. (A) RNA secondary structure prediction was performed usingMfold version 3.1 SEQ. ID. NO. 31. The miRNA sequence is underlined.There is complete homology between mouse and human sequences. (B) Tissueexpression of miR-375 and -376. Total RNA (30 μg) were isolated frommouse tissues for Northern blots and probed for the indicated miRNA. (C)Northern blots of total RNA (10 μg) isolated from purified pancreaticislets, MIN6 cells and total pancreas. High expression levels weredetected in mouse pancreatic islets. A tRNA probe was used as a loadingcontrol.

FIG. 3: Inhibitory action of miR-375 on secretion. (A) MIN6 cells weretransiently co-transfected with 100 ng of plasmid DNA encoding CMV-hGHand β-gal in addition to synthetic siRNAs with homologous sequence tomiRNAs 375, glucokinase or luciferase (si-375, siRNA-Gck and siRNA-luc,respectively) or (B) with 2′-O-methyl-oligoribonucleotides complementaryto miR-375 (2′-O-methyl-375) or a control 2′-O-oligoribonucleotide(2′-O-methyl-GFP). After 48 h, the cells were incubated under low (2.8mM) and stimulatory concentrations of glucose (25 mM). The amount of hGHreleased under these conditions was measured by ELISA and normalized toβ-gal activity. *:P≦0.05, **:P≦0.01).

FIG. 4: Identification of target genes of miR-375. (A) MIN6 cells wereinfected with Ad-miR-375 for 48 h. Following lysis, samples wereseparated by SDS-PAGE, and immunoblotted with α-Mtpn, α-Vti1a, or a-TATAbox binding protein (Tbp) (loading control). (B) Experiment was repeatedusing N2A cells. (C) MIN6 cells transiently transfected with siRNAsdesigned against Mtpn (siRNA-Mtpn) or Vti1a (siVti1a) for 48 h andlysed. After analysis by SDS-PAGE, samples were immunoblotted for eitherMtpn or Vti1a. (D) MIN6 cells were transiently transfected with si-375,si-Mtpn, or si-Vti1a. After 48 h, the cells were incubated under low(2.8 mM) and stimulatory concentrations of glucose (25 mM). The amountof hGH released under these conditions was measured by ELISA andnormalized to β-gal activity. *:P≦0.05, **:P≦0.01).

FIG. 5: The miR-375 target site in the 3′UTR of Mtpn is responsible forinhibition of gene expression by miR-375 SEQ. ID. NO. 1. (A) Sequence ofthe target site in the 3′UTR of myotrophin inserted within the Renillaluciferase 3′ UTR. The mutant construct (Mtpn-MUT) SEQ. ID. NO. 70 isidentical to the WT construct (Mtpn-WT) SEQ. ID. NO. 69 except for fivepoint mutations (bold) disrupting base-pairing at the 5′ end of miR-375.(B) MIN6 cells were transiently transfected with either reporterconstruct in addition to 2′-O-methyl-oligoribonucleotides complementaryto miR-375 (2′-O-methyl-375) or a control 2′-β-oligoribonucleotide(2′-O-methyl-GFP).

DETAILED DESCRIPTION OF THE INVENTION

Pancreatic Islet MicroRNA Molecules

The inventors have discovered novel pancreatic islet microRNA molecules.These molecules have SEQ ID NOs:1-20. Thus, in one embodiment, theinvention relates to an isolated single stranded pancreatic isletmicroRNA molecule.

MicroRNA molecules are known in the art (see, for example, Bartel, Cell,2004, 116, 281-297 for a review on microRNA molecules). The definitionsand characterizations of microRNA molecules in the article by Bartel ishereby incorporated by reference. Such molecules are derived fromgenomic loci and are produced from specific microRNA genes.

Mature microRNA molecules are processed from precursor transcripts thatform local hairpin structures. The hairpin structures are typicallycleaved by an enzyme known as Dicer, generating thereby one microRNAduplex. See the above reference by Bartel.

Usually, one of the two strands of a microRNA duplex is packaged in amicroRNA ribonucleoprotein complex (microRNP). A microRNP in, forexample, humans, also includes the proteins eIF2C2, the helicase Gemin3,and Gemin 4.

In one embodiment, the invention relates to an isolated DNA or RNAmolecule comprising at least ten contiguous bases having a sequenceshown in SEQ ID NOs:1-20, and equivalents thereof. Preferably, theisolated DNA or RNA molecule comprises at least thirteen, morepreferably at least fifteen, and even more preferably at least twentycontiguous bases having a sequence of bases in a pancreatic isletmicroRNA shown in SEQ ID NOs:1-20.

TABLE APancreatic Islet microRNAs and Hairpin Precursor Sequences. The name of the microRNA withthe prefix “hsa” indicates human and the prefix “mmu”indicates a mouse sequence. Thepancreatic islet microRNA sequence portion in the hairpin precursor is indicated in bold.Name MicroRNA (5′ to 3′) Hairpin Precursor (5′ to 3′) hsa-miR-375UUUGUUCGUUCGGCUCGCGUGA CCCCGCGACGAGCCCCUCGCACAAACCGGACCUGAGCGUUUUGUU(Isl-1) (SEQ ID 1) CGUUCGGCUCGCGUGAGGC (SEQ ID NO: 21) hsa-miR-376AUCAUAGAGGAAAAUCCACGU UAAAAGGUAGAUUCUCCUUCUAUGAGUACAUUAUUUAUGAUUAAU(Isl-2) (SEQ ID NO: 2) CAUAGAGGAAAAUCCACGUUUUC (SEQ ID NO: 22)hsa-miR-377 AUCACACAAAGGCAACUUUUGUUUGAGCAGAGGUUGCCCUUGGUGAAUUCGCUUUAUUUAUGUUGAA (SEQ ID NO: 3)UCACACAAAGGCAACUUUUGUUUG (SEQ ID NO: 23) hsa-miR-378CUCCUGACUCCAGGUCCUGUGU GGGGCUCCUGACUCCAGGUCCUGUGUGUUACCUCGAAAUAGCACU(SEQ ID NO: 4) GGACUUGGAGUCAGAAGGCCU (SEQ ID NO: 24) hsa-miR-379UGGUAGACUAUGGAACGUA AGAGAUGGUAGACUAUGGAACGUAGGCGUUAUGAUUUCUGACCUA(SEQ ID NO: 5) UGUAACAUGGUCCACUAACUCU (SEQ ID NO: 25) hsa-miR-380UGGUUGACCAUAGAACAUG AAGAUGGUUGACCAUAGAACAUGCGCUAUCUCUGUGUCGUAUGUA(SEQ ID NO: 6) AUAUGGUCCACAUCUU (SEQ ID NO: 26) hsa-miR-381UAUACAAGGGCAAGCUCUCUGU UACUUAAAGCGAGGUUGCCCUUUGUAUAUUCGGUUUAUUGACAUG(SEQ ID NO: 7) GAAUAUACAAGGGCAAGCUCUCUGUGAGUA (SEQ ID NO: 27)hsa-miR-382 GAAGUUGUUCGUGGUGGAUUCGUACUUGAAGAGAAGUUGUUCGUGGUGGAUUCGCUUUACUUAUGAC (SEQ ID NO: 8)GAAUCAUUCACGGACAACACUUUUUUCAGUA (SEQ ID NO: 28) hsa-miR-383AGAUCAGAAGGUGACUGUGGCU CUCCUCAGAUCAGAAGGUGAUUGUGGCUUUGGGUGGAUAUUAAUC(SEQ ID NO: 9) AGCCACAGCACUGCCUGGUCAGAAAGAG (SEQ ID NO: 29) hsa-miR-384AUUCCUAGAAAUUGUUCAUA UGUUAAAUCAGGAAUUUUAAACAAUUCCUAGACAAUAUGUAUAAU(SEQ ID NO: 10) GUUCAUAAGUCAUUCCUAGAAAUUGUUCAUAAUGCCUGUAACA(SEQ ID NO: 30) mmu-miR-375 UUUGUUCGUUCGGCUCGCGUGACCCCGCGACGAGCCCCUCGCACAAACCGGACCUGAGCGUUUUGUUCG (Isl-1) (SEQ ID NO: 11)UUCGGCUCGCGUGAGGC (SEQ ID NO: 31) mmu-miR-376 AUCGUAGAGGAAAAUCCACGUUAAAAGGUAGAUUCUCCUUCUAUGAGUACAAUAUUAAUGACUAAUCG (Isl-2) (SEQ ID NO: 12)UAGAGGAAAAUCCACGUUUUC (SEQ ID NO: 32) mmu-miR-377 AUCACACAAAGGCAACUUUUGUUGAGCAGAGGUUGCCCUUGGUGAAUUCGCUUUAUUGAUGUUGAAUCA (SEQ ID NO: 13)CACAAAGGCAACUUUUGUUUG (SEQ ID NO: 33) mmu-miR-378 CUCCUGACUCCAGGUCCUGUGUGGGGCUCCUGACUCCAGGUCCUGUGUGUUACCUCGAAAUAGCACUGG (SEQ ID NO: 14)ACUUGGAGUCAGAAGGCCU (SEQ ID NO: 34) mmu-miR-379 UGGUAGACUAUGGAACGUAAGAGAUGGUAGACUAUGGAACGUAGGCGUUAUGUUUUUGACCUAUGU (SEQ ID NO: 15)AACAUGGUCCACUAACUCU (SEQ ID NO: 35) mmu-miR-380 UGGUUGACCAUAGAACAUGAAGAUGGUUGACCAUAGAACAUGCGCUACUUCUGUGUCGUAUGUAGU (SEQ ID NO: 16)AUGGUCCACAUCUU (SEQ ID NO: 36) mmu-miR-381 UAUACAAGGGCAAGCUCUCUGUUACUUAAAGCGAGGUUGCCCUUUGUAUAUUCGGUUUAUUGACAUGGA (SEQ ID NO: 17)AUAUACAAGGGCAAGCUCUCUGUGAGUA (SEQ ID NO: 37) mmu-miR-382GAAGUUGUUCGUGGUGGAUUCG UACUUGAAGAGAAGUUGUUCGUGGUGGAUUCGCUUUACUUGUGACGA(SEQ ID NO: 18) AUCAUUCACGGACAACACUUUUUUCAGUA (SEQ ID NO: 38)mmu-miR-383 AGAUCAGAAGGUGACUGUGGCUCUCAGAUCAGAAGGUGACUGUGGCUUUGGGUGGAUAUUAAUCAGCCA (SEQ ID NO: 19)CAGCACUGCCUGGUCAGAAAGAG (SEQ ID NO: 39) mmu-miR-384 AUUCCUAGAAAUUGUUCACAUGUUAAAUCAGGAAUUGUAAACAAUUCCUAGGCAAUGUGUAUAAUGU (SEQ ID N0:20)UGGUAAGUCAUUCCUAGAAAUUGUUCACAAUGCCUGUAACA (SEQ ID NO: 40)

In this specification, a base refers to any one of the nucleotide basesnormally found in naturally occurring DNA or RNA. The bases can bepurines or pyrimidines. Examples of purine bases include adenine (A) andguanine (G). Examples of pyrimidine bases include thymine (T), cytosine(C) and uracil (U). The adenine can be replaced with 2,6-diaminopurine.

Sequences of unmodified nucleic acid molecules disclosed in thisspecification are shown having uracil bases. Uracil bases occur inunmodified RNA molecules. The invention also includes unmodified DNAmolecules. The sequence of bases of the unmodified DNA molecule is thesame as the unmodified RNA molecule, except that in the unmodified DNAmolecule, the uracil bases are replaced with thymine bases.

Each base in the sequence can form a Watson-Crick base pair with acomplementary base. Watson-Crick base pairs as used herein refer to thehydrogen bonding interaction between, for example, the following bases:adenine and thymine (A-T); adenine and uracil (A-U); and cytosine andguanine (C-G).

Equivalents refer to molecules wherein up to thirty percent of the atleast ten contiguous bases are wobble bases, and up to ten percent, andpreferably up to five percent of the contiguous bases arenon-complementary.

As used herein, wobble base refer to either: 1) substitution of acytosine with a uracil, or 2) the substitution of an adenine with aguanine, in the sequence of the molecule. These wobble basesubstitutions are generally referred to as UG or GU wobbles. Table Bshows the number of contiguous bases and the maximum number of wobblebases in the molecule.

TABLE B Number of contiguous Bases and Maximum Number of Wobble BasesNo. of Contiguous Bases 10 11 12 13 14 15 16 17 18 Max. No. of 3 3 3 3 44 4 5 5 Wobble Base Pairs No. of Contiguous Bases 19 20 21 22 23 Max.No. of 5 6 6 6 6 Wobble Base Pairs

The term “non-complementary” as used herein refers to additions,deletions, mismatches or combinations thereof. Additions refer to theinsertion in the contiguous sequence of any base described above.Deletions refer to the removal of any moiety present in the contiguoussequence. Mismatches refer to the substitution of one of the bases inthe contiguous sequence with a different base.

The additions, deletions or mismatches can occur anywhere in thecontiguous sequence, for example, at either end of the contiguoussequence or within the contiguous sequence of the molecule. Typically,the additions, deletions or mismatches occur at the end of thecontiguous if the contiguous sequence is relatively short, such as, forexample, from about ten to about fifteen bases in length. If thecontiguous sequence is relatively long, such as, for example, a minimumof sixteen contiguous sequences, the additions, deletions, or mismatchesmay occur anywhere in the contiguous sequence.

For example, none or one of the contiguous bases may be additions,deletions, or mismatches when the number of contiguous bases is ten tonineteen; and a maximum of one or two additions, deletions, ormismatches are permissible when the number of contiguous bases is twentyto twenty-three.

In addition to the at least ten contiguous nucleotides of the pancreaticislet microRNA, the isolated DNA or RNA molecule may also have one ormore additional nucleotides. There is no upper limit to the additionalnumber of nucleotides. Typically, no more than about 500 nucleotides,and preferably no more than about 300 nucleotides are added to the atleast ten contiguous bases of a pancreatic islet microRNA.

Any nucleotide can be added. The additional nucleotides can comprise anybase described above. Thus, for example, the additional nucleotides maybe any one or more of A, G, C, T, or U.

In one embodiment, the pancreatic islet microRNA is part of a hairpinprecursor sequence or fragment thereof. For example, suitable hairpinprecursor sequences are shown in SEQ ID NOs:21-40. The fragment can beany fragment of the hairpin precursor sequence containing at least ten,preferably at least fifteen, more preferably at least twenty nucleotidesat the 5′ end and/or nucleotides at the 3′ end. Preferably the sequenceof nucleotides is in the hairpin precursor in which the pancreatic isletmicroRNA is present.

The pancreatic islet microRNA or haipin precursor can be inserted into avector, such as, for example, a recombinant vector. Typically, toconstruct a recombinant vector containing a pancreatic islet microRNA,the hairpin precursor sequence which contains the pancreatic isletmicroRNA sequence is incorporated into the vector. See for example, Chenet al. Science 2004, 303:83-86.

The recombinant vector may be any recombinant vector, such as a plasmid,a cosmid or a phage. Recombinant vectors generally have an origin ofreplication. The vector may be, for example, a viral vector, such as anadenovirus vector or an adeno-associated virus (AAV) vector. See forexample: Ledley 1996, Pharmaceutical Research 13:1595-1614 and Verma etal. Nature 1997, 387:239-242.

The vector may further include a selectable marker. Suitable selectablemarkers include a drug resistance marker, such as tetracycline orgentamycin, or a detectable gene marker, such as β-galactosidase orluciferase.

In a preferred embodiment, the isolated DNA or RNA molecule consistsessentially of any one of the pancreatic islet microRNA sequences or ahairpin precursor sequence shown in SEQ ID NOs:1-40.

In this specification, “isolated” means that the molecule is essentiallyfree of other nucleic acids. Essentially free from other nucleic acidsmeans that the molecule is at least about 90%, preferably at least about95% and, more preferably at least about 98% free of other nucleic acids.

Preferably, the molecule is essentially pure, which means that themolecules are free not only of other nucleic acids, but also of othermaterials used in the synthesis and isolation of the molecule. Materialsused in synthesis include, for example, enzymes. Materials used inisolation include, for example, gels, such as SDS-PAGE. The molecule isat least about 90% free, preferably at least about 95% free and, morepreferably at least about 98% free of such materials.

The islet cells can be any pancreatic islet cell known to those in theart. Examples of pancreatic islet cells include alpha cells, beta cells,delta cells, PP cells and D1 cells. Preferably, the cells are betacells.

The sequence of bases in a microRNA or hairpin precursor is highlyconserved. Due to the high conservation, the sequence can be from apancreatic cell of any mammal. Examples of mammals include pet animals,such as dogs and cats, farm animals, such as cows, horses and sheeps,laboratory animals, such as rats, mice and rabbits, and primates, suchas monkeys and humans. Preferably, the mammal is human or mouse.

Modified Single Stranded Pancreatic Islet MicroRNA Molecules

In another embodiment, the invention relates to a modified singlestranded pancreatic islet microRNA molecule. The modified singlestranded microRNA molecule can be any of the pancreatic microRNAmolecules, hairpin precursor molecules, or equivalents thereof describedabove, except that the modified molecule comprises at least one modifiedmoiety (i.e., at least one moiety is not an unmodifieddeoxyribonucleotide moiety or ribonucleotide moiety). In thisembodiment, the modified pancreatic islet microRNA molecule comprises aminimum number of ten moieties, preferably a minimum of thirteen, morepreferably a minimum of fifteen, even more preferably a minimum ofeighteen, and most preferably a minimum of twenty-one moieties.

The modified pancreatic islet microRNA molecules comprise a maximumnumber of fifty modified moieties, preferably a maximum of forty, morepreferably a maximum of thirty, even more preferably a maximum oftwenty-five, and most preferably a maximum of twenty-three modifiedmoieties. A suitable range of minimum and maximum numbers of modifiedmoieties may be obtained by combining any of the above minima with anyof the above maxima.

Each modified moiety comprises a base bonded to a backbone unit. Thebackbone unit may be any molecular unit that is able to stably bind to abase and to form an oligomeric chain. In this specification, thebackbone units of a modified moiety do not include the backbone unitscommonly found in naturally occurring DNA or RNA molecules.

Such modified pancreatic islet microRNA molecules have increasednuclease resistance. Therefore, the nuclease resistance of the moleculeis increased compared to a sequence containing only unmodifiedribonucleotide moieties, unmodified deoxyribonucleotide moieties orboth. Such modified moieties are well known in the art, and werereviewed, for example, by Kurreck, Eur. J. Biochem. 270, 1628-1644(2003).

The nuclease resisted can be an exonuclease, an endonuclease, or both.The exonuclease can be a 3′→5′ exonuclease or a 5→3′ exonuclease.Examples of 3′→5′ human exonuclease include PNPT1, Werner syndromehelicase, RRP40, RRP41, RRP42, RRP45, and RRP46. Examples of 5→3′exonuclease include XRN2, and FEN1. Examples of endonucleases includeDicer, Drosha, RNase4, Ribonuclease P, Ribonuclease H1, DHP1, ERCC-1 andOGG1. Examples of nucleases which function as both an exonuclease and anendonuclease include APE1 and EXO1.

A modified moiety can occur at any position in the pancreatic isletmicroRNA molecule. For example, to protect pancreatic islet microRNAmolecules against 3→5′ exonucleases, the molecules can have at least onemodified moiety at the 3′ end of the molecule and preferably at leasttwo modified moieties at the 3′ end. If it is desirable to protect themolecule against 5→3′ exonuclease, the pancreatic islet microRNAmolecules can have at least one modified moiety and preferably at leasttwo modified moieties at the 5′ end of the molecule. The pancreaticislet microRNA molecules can also have at least one and preferably atleast two modified moieties between the 5′ and 3′ end of the molecule toincrease resistance of the molecule to endonucleases. Preferably, atleast about 10%, more preferably at least about 25%, even morepreferably at least about 50%, and further more preferably at leastabout 75%, and most preferably at least about 95% of the moieties aremodified. In one embodiment, all of the moieties are modified (e.g.,nuclease resistant).

In one example of a modified pancreatic islet microRNA molecule, themolecule comprises at least one modified deoxyribonucleotide moiety.Suitable modified deoxyribonucleotide moieties are known in the art.Such modified deoxyribonucleotide moieties comprise, for example,phosphorothioate deoxyribose groups as the backbone unit. See structure1 in FIG. 1. A modified pancreatic islet microRNA molecule comprisingphosphorothioate deoxyribonucleotide moieties is generally referred toas phosphorothioate (PS) DNA. See, for example, Eckstein, AntisenseNucleic Acids Drug Dev. 10, 117-121 (2000).

Another suitable example of a modified deoxyribonucleotide moiety is anN′3-N′5 phosphoroamidate deoxyribonucleotide moiety, which comprises anN′3-N′5 phosphoroamidate deoxyribose group as the backbone unit. Seestructure 2 in FIG. 1. An oligonucleotide molecule comprisingphosphoroamidate deoxyribonucleotide moieties is generally referred toas phosphoroamidate (NP) DNA. See, for example, Gryaznov et al., J. Am.Chem. Soc. 116, 3143-3144 (1994).

In another example of a modified pancreatic islet microRNA molecule, themolecule comprises at least one modified ribonucleotide moiety. Asuitable example of a modified ribonucleotide moiety is a ribonucleotidemoiety that is substituted at the 2′ position. The substituents at the2′ position may, for example, be a C₁ to C₄ alkyl group. The C₁ to C₄alkyl group may be saturated or unsaturated, and unbranched or branched.Some examples of C₁ to C₄ alkyl groups include ethyl, isopropyl, andallyl. The preferred C₁ to C₄ alkyl group is methyl. See structure 3 inFIG. 1. An oligoribonucleotide molecule comprising ribonucleotidemoieties substituted at the 2′ position with a C₁ to C₄ alkyl group isgenerally referred to as a 2′-O—(C₁-C₄ alkyl) RNA, e.g., 2′-O-methyl RNA(OMe RNA).

Another suitable example of a substituent at the 2′ position of amodified ribonucleotide moiety is a C₁ to C₄ alkoxy-C₁ to C₄ alkylgroup. The C₁ to C₄ alkoxy (alkyloxy) and C₁ to C₄ alkyl group maycomprise any of the alkyl groups described above. The preferred C₁ to C₄alkoxy-C₁ to C₄ alkyl group is methoxyethyl. See structure 4 in FIG. 1.An oligonucleotide molecule comprising more than one ribonucleotidemoiety that is substituted at the 2′ position with a C₁ to C₄ alkoxy-C₁to C₄ alkyl group is referred to as a 2′-O—(C₁ to C₄ alkoxy-C₁ to C₄alkyl) RNA, e.g., 2′-O-methoxyethyl RNA (MOE RNA).

Another suitable example of a modified ribonucleotide moiety is aribonucleotide that has a methylene bridge between the 2′-oxygen atomand the 4′-carbon atom. See structure 5 in FIG. 1. Anoligoribonucleotide molecule comprising ribonucleotide moieties that hasa methylene bridge between the 2′-oxygen atom and the 4′-carbon atom isgenerally referred to as locked nucleic acid (LNA). See, for example,Kurreck et al., Nucleic Acids Res. 30, 1911-1918 (2002); Elayadi et al.,Curr. Opinion Invest. Drugs 2, 558-561 (2001); Ørum et al., Curr.Opinion Mol. Ther. 3, 239-243 (2001); Koshkin et al., Tetrahedron 54,3607-3630 (1998); Obika et al., Tetrahedron Lett. 39, 5401-5404 (1998).Locked nucleic acids are commercially available from Proligo (Paris,France and Boulder, Colo., USA).

Another suitable example of a modified ribonucleotide moiety is aribonucleotide that is substituted at the 2′ position with fluoro group.Such 2′-fluororibonucleotide moieties are known in the art. Moleculescomprising 2′-fluororibonucleotide moieties are generally referred toherein as 2′-fluororibo nucleic acids (FANA). See structure 7 in FIG. 1.Damha et al., J. Am. Chem. Soc. 120, 12976-12977 (1998).

In another example of a modified pancreatic islet microRNA molecule, themolecule comprises at least one modified moiety comprising a base bondedto an amino acid residue as the backbone unit. Modified moieties thathave at least one base bonded to an amino acid residue will be referredto herein as peptide nucleic acid (PNA) moieties. Such moieties arenuclease resistance, and are known in the art. Molecules having PNAmoieties are generally referred to as peptide nucleic acids. Seestructure 6 in FIG. 1. Nielson, Methods Enzymol. 313, 156-164 (1999);Elayadi, et al, id.; Braasch et al., Biochemistry 41, 4503-4509 (2002),Nielsen et al., Science 254, 1497-1500 (1991).

The amino acids can be any amino acid, including natural or non-naturalamino acids. Naturally occurring amino acids include, for example, thetwenty most common amino acids normally found in proteins, i.e., alanine(Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine(Cys), glutamine (Glu), glutamic acid (Glu), glycine (Gly), histidine(His), isoleucine (Ileu), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan, (Trp), tyrosine (Tyr), and valine (Val).

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups. Some examples of alkyl amino acids includeα-aminobutyric acid, β-aminobutyric acid, γ-aminobutyric acid,δ-aminovaleric acid, and ε-aminocaproic acid. Some examples of arylamino acids include ortho-, meta, and para-aminobenzoic acid. Someexamples of alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.

Non-naturally occurring amino acids also include derivatives ofnaturally occurring amino acids. The derivative of a naturally occurringamino acid may, for example, include the addition or one or morechemical groups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include hydroxyl, C₁-C₄ alkoxy, amino, methylamino,dimethylamino, nitro, halo (i.e., fluoro, chloro, bromo, or iodo), orbranched or unbranched C₁-C₄ alkyl, such as methyl, ethyl, n-propyl,isopropyl, butyl, isobutyl, or t-butyl.

Other examples of non-naturally occurring amino acids which arederivatives of naturally occurring amino acids include norvaline (Nva),norleucine (Nle), and hydroxyproline (Hyp).

The amino acids can be identical or different from one another. Basesare attached to the amino acid unit by molecular linkages. Examples oflinkages are methylene carbonyl, ethylene carbonyl and ethyl linkages.(Nielsen et al., Peptide Nucleic Acids-Protocols and Applications,Horizon Scientific Press, pages 1-19; Nielsen et al., Science 254:1497-1500.) One example of an amino acid residue of a PNA moiety isN-(2-aminoethyl)-glycine.

Further examples of PNA moieties include cyclohexyl PNA, retro-inversoPNA, phosphone PNA, propionyl PNA and aminoproline PNA moieties. For adescription of these PNA moieties, see FIG. 5 of Nielsen et al., PeptideNucleic Acids-Protocols and Applications, Horizon Scientific Press,pages 1-19. FIG. 5 on page 7 of Nielsen et al. is hereby incorporated byreference.

PNA can be chemically synthesized by methods known in the art, e.g. bymodified Fmoc or tBoc peptide synthesis protocols. The PNA has manydesirable properties, including high melting temperatures (Tm), highbase-pairing specificity with nucleic acid and an uncharged molecularbackbone. Additionally, the PNA does not confer RNase H sensitivity onthe target RNA, and generally has good metabolic stability.

Peptide nucleic acids are also commercially available from AppliedBiosystems (Foster City, Calif., USA).

In another example of a modified pancreatic islet microRNA molecule, themolecule comprises at least one morpholino phosphoroamidate nucleotidemoiety. Molecules comprising morpholino phosphoroamidate nucleotidemoieties are generally referred to as morpholino (MF) nucleic acids. Seestructure 8 in FIG. 1. Heasman, Dev. Biol. 243, 209-214 (2002).Morpholino oligonucleotides are commercially available from Gene ToolsLLC (Corvallis, Oreg., USA).

In a further example of a modified pancreatic islet microRNA molecule,the molecule comprises at least one cyclohexene nucleotide moiety.Molecules comprising cyclohexene nucleotide moieties are generallyreferred to as cyclohexene nucleic acids (CeNA). See structure 10 inFIG. 1. Wang et al., J. Am. Chem. Soc. 122, 8595-8602 (2000), Verbeureet al., Nucleic Acids Res. 29, 4941-4947 (2001).

In a final example of a modified pancreatic islet microRNA molecule, themolecule comprises at least one tricyclo nucleotide moiety. Moleculescomprising tricyclo nucleotide moieties are generally referred to astricyclo nucleic acids (tcDNA). See structure 9 in FIG. 1. Steffens etal., J. Am. Chem. Soc. 119, 11548-11549 (1997), Renneberg et al., J. Am.Chem. Soc. 124, 5993-6002 (2002).

Chimeric pancreatic modified islet microRNA molecules containing amixture of any of the moieties mentioned above are also known, and maybe made by methods known, in the art.

See, for example, references cited above, and Wang et al, Proc. Natl.Acad. Sci. USA 96, 13989-13994 (1999), Liang et al., Eur. J. Biochem.269, 5753-5758 (2002), Lok et al., Biochemistry 41, 3457-3467 (2002),and Damha et al., J. Am. Chem. Soc. 120, 12976-12977 (2002).

The modified pancreatic islet microRNA molecules of the inventioncomprise at least ten, preferably at least thirteen, more preferably atleast fifteen, and even more preferably at least twenty contiguous baseshaving any of the contiguous base sequences of a naturally occurringpancreatic islet microRNA molecule shown in SEQ ID NOs:1-20. In apreferred embodiment, the modified pancreatic islet microRNA moleculescomprise the entire sequence of any of the pancreatic islet microRNAmolecule shown in SEQ ID NOs:1-20.

Any number of additional moieties, up to a maximum of forty moieties,having any base sequence can be added to the moieties comprising thecontiguous base sequence, as long as the total number of moieties in themolecule does not exceed fifty. The additional moieties can be added tothe 5′ end, the 3′ end, or to both ends of the contiguous sequence. Theadditional moieties can include a sequence of bases at the 5′ end and/ora sequence of bases at the 3′ end present in the hairpin precursor fromwhich the pancreatic islet microRNA is derived or any fragment thereof.The additional moieties in the molecule, if any, can be any modified orunmodified moiety described above.

The modified pancreatic islet microRNA molecules include equivalentsthereof. Equivalents include wobble bases and non-complementary bases asdescribed above.

Further, no more than fifty percent, and preferably no more than thirtypercent, of the contiguous moieties contain deoxyribonucleotide backboneunits. Table C and D show the maximum number of deoxyribonucleotidebackbone units for each number of contiguous bases.

In another embodiment, in addition to the wobble base pairs andnon-complementary bases described above, the moiety corresponding toposition 11 in a naturally occurring pancreatic islet microRNA sequencecan be an addition, deletion or mismatch.

The modified pancreatic islet microRNA molecule is preferably isolated,more preferably purified, as defined above.

TABLE C Fifty Percent of the Contiguous Moieties containingDeoxyribonucleotide Backbone Units No. of Contiguous Bases 10 11 12 1314 15 16 17 18 Max. No. of 5 5 6 6 7 7 8 8 9 DeoxyribonucleotideBackbone Units No. of Contiguous Bases 19 20 21 22 23 Max. No. of 9 1010 11 11 Deoxyribonucleotide Backbone Units

TABLE D Thirty Percent of the Contiguous Moieties ContainingDeoxyribonucleotide Backbone Units No. of Contiguous Bases 10 11 12 1314 15 16 17 18 Max. No. of 3 3 3 3 4 4 4 5 5 DeoxyribonucleotideBackbone Units No. of Contiguous Bases 19 20 21 22 23 Max. No. of 5 6 66 6 Deoxyribonucleotide Backbone Units

In yet another embodiment, caps can be attached to one end, both ends,and/or between the ends of the molecule in order to increase nucleaseresistance of the modified pancreatic islet microRNA molecules orunmodified isolated DNA or RNA molecules of the present inventiondescribed above to exonucleases. Any cap known to those in the art forincreasing nuclease resistance can be employed. Examples of such capsinclude inverted nucleotide caps and chemical caps.

An inverted nucleotide cap refers to a 3′→5′ sequence of nucleic acidsattached to the pancreatic islet microRNA molecule at the 5′ and/or the3′ end. There is no limit to the maximum number of nucleotides in theinverted cap just as long as it does not interfere with binding of thepancreatic islet microRNA molecule or isolated DNA or RNA molecule toits target mRNA. Any nucleotide can be used in the inverted nucleotidecap. Usually, the nucleotide cap is less than about forty nucleotides inlength, preferably less than about thirty nucleotides in length, morepreferably less than about twenty nucleotides in length, and even morepreferably less than about ten nucleotides in length. Typically, theinverted nucleotide cap is one nucleotide in length. The nucleotide forthe inverted cap is generally thymine, but can be any nucleotide such asadenine, guanine, uracil, or cytosine.

Alternatively, a chemical cap can be attached to the 5′ end, to the 3′end, to both ends of the molecule, and/or to any moiety(ies) between the5′ end and 3′ end of the modified pancreatic islet microRNA molecule orisolated DNA or RNA molecule in order to increase nuclease resistance toexonucleases and/or endonucleases. The chemical cap can be any chemicalgroup known to those in the art for increasing nuclease resistance ofnucleic acids. Examples of such chemical caps include hydroxyalkyl oraminoalkyl groups. Hydroxyalkyl groups are sometimes referred to asalkyl glycoyl groups (e.g., ethylene glycol). Aminoalkyl groups aresometimes referred to as amino linkers.

The alkyl chain in the hydroxyalkyl group or aminoalkyl groups can be astraight chain or branched chain. The minimum number of carbon atomspresent in the alkyl chain is one, preferably at least two, and morepreferably at least about three carbon atoms. The maximum number ofcarbon atoms present in the alkyl chain is about eighteen, preferablyabout sixteen, and more preferably about twelve. Typical alkyl groupsinclude methyl, ethyl, and propyl. The alkyl groups can be furthersubstituted with one or more hydroxyl and/or amino groups.

Some examples of amino linkers are shown in Table E. The amino linkerslisted in Table E are commercially available from TriLinkBiotechnologies, San Diego, Calif.

Isolated MicroRNP

In another aspect, the invention provides an isolated microRNPcomprising any of the isolated DNA or RNA molecules described above ormodified pancreatic islet microRNA molecules described above.

Anti-Pancreatic Islet MicroRNA Molecules

In another aspect, the invention provides an anti-pancreatic isletmicroRNA molecule.

The anti-pancreatic islet microRNA molecule may be any of the isolatedDNA or RNA molecules described above or modified pancreatic isletmicroRNA molecules described above, except that the sequence of bases ofthe anti-pancreatic islet microRNA molecule is complementary to thesequence of bases in an isolated DNA or RNA molecule or modifiedpancreatic islet microRNA molecule.

Examples of sequences of anti-pancreatic islet microRNA molecules areshown in Table F.

TABLE E Amino Linkers from TriLink Biotechnologies 2′-Deoxycytidine-5-C6Amino Linker (3′ Terminus) 2′-Deoxycytidine-5-C6 Amino Linker (5′ orInternal) 3′ C3 Amino Linker 3′ C6 Amino Linker 3′ C7 Amino Linker 5′C12 Amino Linker 5′ C3 Amino Linker 5′ C6 Amino Linker C7 Internal AminoLinker Thymidine-5-C2 Amino Linker (5′ or Internal) Thymidine-5-C6 AminoLinker (3′ Terminus) Thymidine-5-C6 Amino Linker (Internal)

TABLE F Anti-pancreatic islet microRNA Sequences MicroRNAAnti-microRNA Sequence (5′ → 3′) hsa-miR-375 UCACGCGAGCCGAACGAACAAA(Isl-1) (SEQ ID NO: 41) hsa-miR-376 ACGUGGAUUUUCCUCUAUGAU (Isl-2)(SEQ ID NO: 42) hsa-miR-377 ACAAAAGUUGCCUUUGUGUGAU (SEQ ID NO: 43)hsa-miR-378 ACACAGGACCUGGAGUCAGGAG (SEQ ID NO: 44) hsa-miR-379UACGUUCCAUAGUCUACCA (SEQ ID NO: 45) hsa-miR-380 CAUGUUCUAUGGUCAACCA(SEQ ID NO: 46) hsa-miR-381 ACAGAGAGCUUGCCCUUGUAUA (SEQ ID NO: 47)hsa-miR-382 CGAAUCCACCACGAACAACUUC (SEQ ID NO: 48) hsa-miR-383AGCCACAAUCACCUUCUGAUCU (SEQ ID NO: 49) hsa-miR-384 UAUGAACAAUUUCUAGGAAU(SEQ ID NO: 50) mmu-miR-375 UCACGCGAGCCGAACGAACAAA (Isl-1)(SEQ ID NO: 51) mmu-miR-376 ACGUGGAUUUUCCUCUACGAU (Isl-2)(SEQ ID NO: 52) mmu-miR-377 ACAAAAGUUGCCUUUGUGUGAU (SEQ ID NO: 53)mmu-miR-378 ACACAGGACCUGGAGUCAGGAG (SEQ ID NO: 54) mmu-miR-379UACGUUCCAUAGUCUACCA (SEQ ID NO: 55) mmu-miR-380 CAUGUUCUAUGGUCAACCA(SEQ ID NO: 56) mmu-miR-381 ACAGAGAGCUUGCCCUUGUAUA (SEQ ID NO: 57)mmu-miR-382 CGAAUCCACCACGAACAACUUC (SEQ ID NO: 58) mmu-miR-383AGCCACAGUCACCUUCUGAUCU (SEQ ID NO: 59) mmu-miR-384 UGUGAACAAUUUCUAGGAAU(SEQ ID NO: 60)

The anti-pancreatic islet microRNA molecule can be modified as describedabove for modified pancreatic islet microRNA molecules. In oneembodiment, the contiguous moieties in the anti-pancreatic isletmicroRNA molecule are complementary to the corresponding pancreaticislet microRNA molecule. The degree of complementarity of theanti-pancreatic islet microRNA molecules are subject to the samerestrictions described above for modified pancreatic islet microRNAmolecules, including the restriction relating to wobble base pairs, aswell as those relating to additions, deletions and mismatches.

In a preferable embodiment, if the anti-micro pancreatic microRNAmolecule comprises only unmodified moieties, then the anti-pancreaticislet microRNA molecule comprises at least one base, in the at least tencontiguous bases, which is non-complementary to the pancreatic isletmicroRNA and/or comprises a chemical cap.

In another preferable embodiment, if the at least ten contiguous basesin an anti-pancreatic islet microRNA molecule is perfectly (i.e., 100%)complementary to a pancreatic islet microRNA molecule, then theanti-pancreatic islet microRNA molecule contains at least one modifiedmoiety in the at least ten contiguous bases and/or comprises a chemicalcap.

In yet another embodiment, the moiety in the anti-pancreatic isletmicroRNA molecule at the position corresponding to position 11 of anaturally occurring pancreatic islet microRNA is non-complementary. Themoiety in the anti-pancreatic islet microRNA molecule corresponding toposition 11 of a naturally occurring pancreatic islet microRNA can berendered non-complementary by the introduction of an addition, deletionor mismatch, as described above.

Utility

The pancreatic islet microRNA molecules and anti-pancreatic isletmicroRNA molecules of the present invention have numerous in vitro, exvivo, and in vivo applications.

For example, the microRNA molecules and/or anti-microRNA molecules ofthe present invention can be introduced into a cell to study thefunction of the microRNA. Any pancreatic islet microRNA molecule and/oranti-pancreatic islet microRNA mentioned above can be introduced into acell for studying their function.

In one embodiment, a microRNA in a cell is inhibited with a suitableanti-pancreatic islet microRNA molecule. Alternatively, the activity ofa pancreatic islet microRNA molecule in a cell can be enhanced byintroducing into the cell an additional microRNA molecule. The functionof the microRNA can be inferred by observing changes associated withinhibition and/or enhanced activity of the microRNA in the cell.

Thus, in one aspect of the invention, the invention relates to a methodfor inhibiting microRNP activity in a cell. The microRNP comprises apancreatic microRNA molecule. Any anti-pancreatic islet microRNAmolecule can be used in the method for inhibiting microRNP activity in acell, as long as the anti-pancreatic islet microRNA molecule iscomplementary, subject to the restrictions described above, to thepancreatic islet microRNA present in the microRNP.

The anti-pancreatic islet microRNA molecules of the present inventionare capable of inhibiting microRNP activity by binding to the pancreaticislet microRNA in the microRNP in a host cell. MicroRNP activity refersto the cleavage or the repression of translation of a target sequence.The target sequence may be any sequence which is partially or perfectlycomplementary to the sequence of bases in a pancreatic islet microRNA.The target sequence may, for example, be a gene which controls glucoseutilization.

For example, pancreatic islet cells can produce a microRNA which iscomplementary to a gene involved in glucose-induced insulin secretion.The microRNA molecule, which is packaged in a microRNP, will inhibit thebeneficial effect of glucose-induced insulin secretion. Accordingly, theintroduction of the anti-microRNA molecule inhibits the microRNPactivity, and thereby reduces the harm by restoring the function of thegene.

Alternatively, instead of introducing the anti-microRNA moleculementioned above, additional microRNA molecules can be introduced intothe pancreatic islet cell. Accordingly, the gene for glucose-inducedinsulin secretion will be inhibited, thereby decreasing the ability ofthe cell to secrete insulin in response to glucose.

The microRNA molecules and/or anti-microRNA molecules can be introducedinto a cell by any method known to those skilled in the art. The methodfor inhibiting microRNP activity in a cell comprises introducing intothe cell a single-stranded anti-pancreatic islet microRNA molecule.

For example, the microRNA molecules and/or anti-microRNA molecules canbe injected directly into a cell, such as by microinjection.Alternatively, the molecules can be contacted with a cell, preferablyaided by a delivery system.

Useful delivery systems include, for example, liposomes and chargedlipids. Liposomes typically encapsulate oligonucleotide molecules withintheir aqueous center. Charged lipids generally formlipid-oligonucleotide molecule complexes as a result of opposingcharges.

These liposomes-oligonucleotide molecule complexes orlipid-oligonucleotide molecule complexes are usually internalized incells by endocytosis. The liposomes or charged lipids generally comprisehelper lipids which disrupt the endosomal membrane and release theoligonucleotide molecules.

Other methods for introducing a microRNA molecule or an anti-microRNAinto a cell include use of delivery vehicles, such as dendrimers,biodegradable polymers, polymers of amino acids, polymers of sugars, andoligonucleotide-binding nanoparticles. In addition, pluoronic gel as adepot reservoir can be used to deliver the anti-microRNA oligonucleotidemolecules over a prolonged period. The above methods are described in,for example, Hughes et al., Drug Discovery Today 6, 303-315 (2001);Liang et al. Eur. J. Biochem. 269 5753-5758 (2002); and Becker et al.,In Antisense Technology in the Central Nervous System (Leslie, R. A.,Hunter, A. J. & Robertson, H. A., eds), pp. 147-157, Oxford UniversityPress.

Targeting of a microRNA molecule or an anti-microRNA molecule to aparticular cell can be performed by any method known to those skilled inthe art. For example, the microRNA molecule or anti-microRNA moleculecan be conjugated to an antibody or ligand specifically recognized byreceptors on the cell. For example, the ligand can be GLP-1(glucagons-like peptide) which binds GLP-receptors expressed onpancreatic beta-cells. Alternatively, an antibody to GLP-1 can beemployed.

In another embodiment, the invention provides a method for treatingdiabetes in a mammal in need thereof. The method comprises introducinginto the mammal an effective amount of an anti-pancreatic islet microRNAmolecule having at least ten contiguous bases having a sequence shown inSEQ ID NOs:41 or 51. The effective amount is determined duringpre-clinical trials and clinical trials by methods familiar tophysicians and clinicians.

The anti-pancreatic islet microRNA molecules can be introduced into themammal by any method known to those in the art. For example, the abovedescribed methods for introducing the anti-pancreatic islet moleculesinto a cell can also be used for introducing the molecules into amammal.

The molecules can be administered to a mammal by any method known tothose skilled in the art. Some examples of suitable modes ofadministration include oral and systemic administration. Systemicadministration can be enteral or parenteral. Liquid or solid (e.g.,tablets, gelatin capsules) formulations can be employed.

Parenteral administration of the molecules include, for exampleintravenous, intramuscular, and subcutaneous injections. For instance, amolecule may be administered to a mammal by sustained release, as isknown in the art. Sustained release administration is a method of drugdelivery to achieve a certain level of the drug over a particular periodof time.

Other routes of administration include oral, topical, intrabronchial, orintranasal administration. For oral administration, liquid or solidformulations may be used. Some examples of formulations suitable fororal administration include tablets, gelatin capsules, pills, troches,elixirs, suspensions, syrups, and wafers. Intrabronchial administrationcan include an inhaler spray. For intranasal administration,administration of a molecule of the present invention can beaccomplished by a nebulizer or liquid mist.

The molecules of the present invention can be in a suitablepharmaceutical carrier. In this specification, a pharmaceutical carrieris considered to be synonymous with a vehicle or an excipient as isunderstood by practitioners in the art. Examples of carriers includestarch, milk, sugar, certain types of clay, gelatin, stearic acid orsalts thereof, magnesium or calcium stearate, talc, vegetable fats oroils, gums and glycols.

The pharmaceutical carrier may also comprise one or more of thefollowing: a stabilizer, a surfactant, preferably a nonionic surfactant,and optionally a salt and/or a buffering agent.

The stabilizer may, for example, be an amino acid, such as for instance,glycine; or an oligosaccharide, such as for example, sucrose, tetralose,lactose or a dextran. Alternatively, the stabilizer may be a sugaralcohol, such as for instance, mannitol; or a combination thereof.Preferably the stabilizer or combination of stabilizers constitutes fromabout 0.1% to about 10% weight for weight of the molecules.

The surfactant is preferably a nonionic surfactant, such as apolysorbate. Some examples of suitable surfactants include Tween 20,Tween 80; a polyethylene glycol or a polyoxyethylene polyoxypropyleneglycol, such as Pluronic F-68 at from about 0.001% (w/v) to about 10%(w/v).

The salt or buffering agent may be any salt or buffering agent, such asfor example sodium chloride, or sodium/potassium phosphate,respectively. Preferably, the buffering agent maintains the pH of themolecules of the present invention in the range of about 5.5 to about7.5. The salt and/or buffering agent is also useful to maintain theosmolality at a level suitable for administration to a mammal.Preferably the salt or buffering agent is present at a roughly isotonicconcentration of about 150 mM to about 300 mM.

The pharmaceutical carrier may additionally contain one or moreconventional additives. Some examples of such additives include asolubilizer such as, for example, glycerol; an antioxidant such as forexample, benzalkonium chloride (a mixture of quaternary ammoniumcompounds, known as “quart”), benzyl alcohol, chloretone orchlorobutanol; anaesthetic agent such as for example a morphinederivative; or an isotonic agent etc., such as described above. As afurther precaution against oxidation or other spoilage, the moleculesmay be stored under nitrogen gas in vials sealed with impermeablestoppers.

EXAMPLES Example 1 Materials and Methods

MicroRNA Cloning and Northern Blotting Analysis:

600 μg of total RNA was isolated from MING cell cultures using TRIZOLreagent (Invitrogen) and miRNA cloning was performed as previouslydescribed (Lagos-Quintana, Current Biol.). Antisense probes weredesigned to complement cloned miRNA sequences and used for Northern blotanalysis as previously described (Lagos-Quintana, Current Biol.).

Cell Culture:

MIN6 cells were cultured with DMEM medium containing 25 mM glucose, 15%fetal bovine serum, and 5.5 μM 2-mercaptoethanol. N2A cells werecultured with DMEM medium containing 25 mM glucose and 10% fetal bovineserum.

Insulin Secretion Studies:

MIN6 cells were cultured in 24-well plates for 2 days and washed with amodified Krebs-Ringer buffer (KRBH) (0.9 mM CaCl₂, 2.68 mM KCl, 1.46 mMKH₂PO₄, 0.5 mM MgCl₂.6H₂O, 135 mM NaCl, 8 mM Na₂HPO₄×7H₂O, 20 mM Hepes,and 0.2% BSA) prior to the assay. After a 30 minute pre-incubation withKRBH containing 5.5 mM glucose, cells were rinsed and incubated for 60minutes in KRBH with either 2.8 mM glucose, 25 mM glucose, 30 mM KCl,500 mM tolbutamide, or 5 mM methyl pyruvate. The concentration ofinsulin in the supernatant was measured using RIA (Linco Research).

Generation of Recombinant Adenovirus

The recombinant adenovirus used to overexpress miR-375 was generated byPCR amplifying the miRNA precursor sequence with primers:5′-CCCCAAGGCTGATGCTGAGAAGCCGCCCC-3′ SEQ. ID. NO. 67 and5′-GCCGCCCGGCCCCG GGTCTTC-3′ SEQ. ID. NO. 68. The fragment was subclonedinto pcDNA 3 (Invitrogen), excised with HindIII and XbaI and insertedinto a Ad5CMV-K NpA shuttle vector. Amplification of the adenovirus wasperformed by Viraquest Inc. (North Liberty, Iowa). Ad-GFP (ViraQuestInc.) does not contain a transgene and was used as control.

Electrophysiology and Ca²⁺-Measurements:

Measurements of exocytosis and inward Ca²⁺-currents were conducted onsingle dispersed B-cells ≧24 h after infection with control-GFP- ormiRNA208-GFP-containing adenoviruses using the standard whole-cellconfiguration of the patch-clamp technique. Exocytosis was detected aschanges in cell capacitance, using the software-based lock-inimplementation of Pulse (Heka Electronics. Lamprecht/Pfalz, Germany).The applied sine wave had a frequency of 500 Hz and a peak amplitude of20 mV. The Ca²⁺-currents were measured after leak currents andcapacitive transients had been removed digitally using a −p/4 protocol.The extracellular solution contained (in mM) 118 NaCl, 20 mMtetraethylammonium-chloride (TEA-C1), 5.6 KCl, 2.6 CaCl₂, 1.2 MgCl₂, 5HEPES (pH=7.4) with 5 glucose. In the experiments in which exocytosiswas triggered by voltage-clamp depolarizations, the intracellularsolution consisted of (in mM) 125 Cs-glutamate, 10 CsCl, 10 NaCl, 1MgCl₂, 5 HEPES (pH=7.15 with CsOH), 0.05 EGTA, 3 Mg-ATP and 0.1 cyclicAMP. In one series of experiments exocytosis was evoked dialyzing thecell interior with a medium composed of (in mM) 125 Cs-glutamate, 10KCl, 10 NaCl, 1 MgCl₂, 3 Mg-ATP, 0.1 cAMP, 10 HEPES, 10 EGTA and 9CaCl₂. The free Ca²⁺-concentration of this solution was estimated to be1.5 μM. The experiments were conducted on functionally identified α- andβ-cells. The identity of the cells was established as describedpreviously.

The free intracellular Ca²⁺-concentration ([Ca²⁺]_(i)) was measured bydual-excitation wavelength spectrofluorimetry as described elsewhere.Transfected islets were loaded with 3 μM fura-2 in the presence of0.007% w/v pluronic acid (Molecular probes) for 40 min at 37° C. The dyewas excited at 350 nm and 365 nm. The latter wavelength was used insteadof 380 nm in order to avoid excitation of GFP. Emitted light wascollected at 510 nm. During the experiments the islets were held inplace by a holding pipette and superfused continuously with a mediumcontaining (in mM) 140 NaCl, 3.6 KCl, 2 NaHCO₃, 0.5 NaH₂PO₄, 0.5 MgSO₄,2.6 CaCl₂, 5 HEPES (pH=7.4 mM with NaOH) and 5 mM glucose. The glucoseconcentration was increased to 15 mM and the sulphonylurea tolbutamideadded at a concentration of 0.1 mM as indicated. When the islets weredepolarized with high extracellular K⁺ (30 mM KCl added), theconcentration of NaCl was correspondingly decreased to maintainiso-osmolarity. All electrophysiological experiments andCa²⁺-measurements were carried out at 32-34° C.

The infection of the islets and loading with the Ca²⁺-indicator wereevaluated using confocal microscopy and using fluo-3 instead of fura-2.Excitation of both eGFP and fluo-3 was performed using the 488 nm lineof a Zeiss LSM510 microscope (Carl Zeiss, Jena, Germany). Emitted lightwas separated by using the META facility of the confocal microscope andvisualized using a 40× water objective.

Assay of Luciferase Activity:

The wildtype mouse myotrophin 3′ UTR target site was PCR amplified usingthe following primers: 5′ TCCATCATTTCATATGCACTGTATC 3′ SEQ. ID. NO. 61and 5′ TCATATCGTTAAGGACGTCTGGAAA 3′ SEQ. ID. NO. 62 and subcloned intopCR 2.1 TOPO (Invitrogen). The fragment was removed with SpeI and XbaIand subsequently subcloned into the XbaI site immediately downstream ofstop codon in pRL-TK (Promega). This construct was used to generate themutant mouse myotrophin plasmid using primers: 5′AAGTTTCGTGTTGCAAGCCCCCCTGGAATAAACTTGAATTGTGC 3′ SEQ. ID. NO. 63 and 5′GCACAATTCAAGTTTATTCCAGGGGGGCTTGCAACACGAAACTT 3′ SEQ. ID. NO. 64according to protocol (Stratagene); bold and underline indicate mutatednucleotides. MIN6 cells were cultured in 24 well plates for 2 days andtransfected with both 0.4 μg of the pRL-TK reporter vector coding forRr-luc and 0.1 μg of the pGL3 control vector coding for Pp-luc(Promega). Cells were harvested 30-36 hours post-transfection andassayed.

Identification of miR-375 Targets:

To identify targets of miR-375 we used a recently developed algorithm[N. Rajewsky and N. D. Socci, Developmental Biology 267, 529-535(2004)]. The algorithm consists of two steps: (a) the search for aGC-rich string of consecutive complementary bases (“nucleus”) betweenthe microRNA and the putative target sequence in the 3′ UTRs of mRNAsand (b) in silico evaluation of the free energy of the predictedmicroRNA:mRNA duplex. We applied the algorithm to the Refseq data set.The 3′ UTRs were extracted from the Refseq annotation. This datasetcomprises 18199 human and 13371 mouse 3′ UTRs with a length of at least30 nucleotides. We further used the Jackson lab orthology table of 9612human/mouse orthologs to construct a set of orthologous 3′ UTRs.Following [Lewis B P, Shih I H, Jones-Rhoades M W, Bartel D P, Burge CB, Prediction of mammalian microRNA targets. Cell 787-98 (2003)] werestricted the position of the nucleus to be within 2 bases from the 5′end of the microRNA. The cutoff for the nucleus score in (a) was setsuch that that the top 8% of hits were retained. These hits were thenscored by their predicted mRNA:miRNA duplex free energy via MFOLD(Zuker, NAR 3406-15, 2003; see Rajewsky and Socci for details).

siRNA and 2′-O-methyl Oligoribonucleotides:

Synthetic microRNA and siRNAs were synthesized by Dharmacon Research(Lafayette, Colo.). siRNA SMARTPOOLs were designed from the mousemyotrophin (NM_(—)008098) and mouse Vti1A (NM_(—)016862) sequences. All2′-O-methyl oligoribonucleotides were synthesized as previouslydescribed (Meister et al., RNA). All reagents were tranfected into MIN6cells using Lipofectamine 2000 (Invitrogen) at a 200 nM concentration.

Antibodies:

Antibodies for Western blotting were obtained from several differentsources: α-myotrophin (donated by Masato Taoka), α-Vti1a (BDTransduction Laboratories), α-p38 MAPK (Cell Signaling), α-MCT8 (donatedby Andrew Halestrap), α-TATA box binding protein (donated by R. Roeder).

Northern Blotting:

Total RNA was extracted using TRIZOL reagent (Invitrogen) and loadedonto 15% polyacrylamine or agarose gels. After electrophoresis, RNA wastransferred to Hybond membrane (Amersham) and probed. A DNA probe formouse myotrophin was generated using primers: 5′GTGGGCCCTGAAAAACGGAGACTTG 3′ SEQ. ID. NO. 65 and 5′CCCTTTGACAGAAGCAATTTCACGC 3′ SEQ. ID. NO. 66.

Example 2 Pancreatic Islet MicroRNAs

MicroRNAs from MIN6 cells, a glucose responsive murine pancreatic β-cellline were cloned. We obtained a total of 301 microRNAs clones, whichcontained 55 different microRNAs. Of the 55 different microRNAs, 92%represented previously identified microRNAs and 8% were as-yetunidentified microNAs. Known and novel miRNAs were identified in variousgenome databases by Blast sequence analysis and confirmed bycross-species homology and their ability to form typical hairpinprecursor structures.

A total of 9 novel microRNAs were identified and a single microRNA(miR-375) represented >50% of all novel clones (FIG. 2 a). We nextinvestigated the expression of novel microRNAs by Northern blotanalysis. Only microRNAs 375 and 376 could be detected by Northern blotanalysis from MIN6 cells and pancreatic islets (FIG. 2 b). Theexpression of both microRNAs was restricted to MIN6 cells and pancreaticislets and not found in other tissues including liver, lung, intestine,brain, kidney and testes (FIG. 2 b, c). These data suggested that we hadidentified novel, pancreatic islet microRNAs.

Example 3 Inhibitory Action of miR-375 on Secretion

To analyze the function of the microRNAs with high expression levels andrelative tissue specificity for pancreatic β-cells, we tested the effectof synthetic siRNAs with homologous sequence to microRNAs-375 and -376on glucose-induced insulin secretion in MIN6 cell followingtransfection. In addition to the siRNAs, we cotranfected a vectorexpressing the human growth hormone (hGH) gene under the control of aCMV promoter (CMV-hGH). Exogenously expressed hGH has been previouslyshown to be targeted to secretory granules of pancreatic b-cell linesand to be co-released with insulin after triggering of exocytosis. Thisapproach allowed us to monitor exocytosis selectively from transientlytransfected MIN6 cells (transfection efficiency 20-30%). As positive andnegative controls, siRNAs targeting the glucokinase gene (Gck) orapolipoprotein M (apoM), a gene not expressed in pancreatic β-cells,were cotranfected with CMV-hGH into MIN6 cells.

Growth hormone secretion in response to a 25 mM glucose stimulus wassignificantly decreased in cells transfected with si-Gck and si-375(FIG. 3). Transfection of synthetic siRNA directed against apoM orsiRNAs homologous to several other microRNAs, including miR-376,miR-124, -129, -130, and -210 had no effect on basal orglucose-stimulated insulin secretion (FIG. 3, data not shown).

Antisense-based strategies have recently been shown to specificallyinhibit miRNA function in cultured cells. We co-transfectednuclease-resistant 2′-O-methyl antisense oligoribonucleotides to miR-375with vector CMV-hGH and measured insulin secretion in response tostimulation with glucose. We noted an increase in glucose-stimulatedinsulin secretion of cells transfected with anti-miR-375 compared to acontrol anti-GFP 2′-O-methyl oligoribonucleotide (FIG. 3 b). Together,these data indicated that miR-375 is an inhibitor of insulin secretion.

We next generated a recombinant adenovirus expressing miR-375 by cloninga 123 bp fragment containing the precursor sequence under the control ofthe CMV5 promoter. HEK cells were infected with a control adenovirusexpressing eGFP (Ad-GFP) or increasing concentrations of Ad-375particles showed a dose dependent increase of miR-375 expression. Wealso expressed miR-375 in MIN6 cells using adenoviral infections. MIN6cells expressing miR-375 exhibited a 40% reduction in glucose-inducedinsulin secretion compared to cells that were infected with a controladenovirus (FIG. 4.). The defect in insulin secretion did not appear tobe caused by defective insulin production, since insulin content wasequivalent in Ad-375 and Ad-eGFP infected MIN6 cells and pancreaticislets.

Example 4 Intracellular Calcium Signalling and Whole-Cell CalciumCurrents

An increase in glycolytic flux and mitochondrial oxidativephosphorylation are required to generate secondary signals forglucose-induced insulin secretion by inducing closure of ATP-sensitiveK⁺ channels (KATP) via an increase in the cytosolic ATP/ADP ratio. Thisleads to membrane depolarization and influx of Ca⁺⁺ throughvoltage-dependent Ca⁺⁺ channels. The elevation in [Ca⁺⁺]_(I) is anecessary prerequisite for insulin granule exocytosis. Sulphonylureadrugs like glibenclamide stimulate insulin secretion by directlyblocking KATP. KCl leads to direct depolarization of pancreatic β-cellsand leads to a maximum degranulation of competent insulin granules. Wemeasured insulin secretion in Ad-375 infected MIN6 cells that werestimulated with tolbutamide and KCl. Stimulation of pancreatic β-cellscells with these secretagogues showed impaired insulin secretion inAd-375 infected cells compared to Ad-eGFP infected cells. Furthermore,Ad-375 expression also impaired insulin secretion in response to GLP-1,a potent glucose-dependent stimulator of insulin secretion throughactivation of camp, compared to Ad-eGFP infected MIN6 cells. These datasuggested that overexpression of miR-375 led to a defect that involvesthe distal steps of insulin secretion, possibly affecting a rise incytoplasmic Ca⁺⁺ in pancreatic β-cells or interfering with exocytosis.

To examine whether miR-375 impairs the generation of secondary signalsthat are required to trigger insulin exocytosis, we measuredintracellular Ca⁺⁺ concentrations [Ca²⁺]_(i) in islets that wereinfected with Ad-375 or control Ad-eGFP. Each islet was stimulated bythree different stimuli to increase the intracellularCa²⁺-concentration. Increasing the glucose concentration from 5 mM to 15mM generated oscillations in the Ca²⁺-concentration in both the controland the Ad-375 expressing islets. Similar oscillations were observedwhen stimulating with tolbutamide. The resting [Ca²⁺]_(i) averaged0.13±0.01 μM and 0.12±0.01 μM in control and Ad-375 expressing islets,respectively. The time-averaged [Ca²⁺]_(i) in the presence of 15 mMglucose amounted to 0.42±0.12 μM in control islets and 0.39±0.05 μM inislets expressing Ad-375. The corresponding values in the presence oftolbutamide were 0.53±0.11 μM in the control islets and 0.55±0.11 μM inthe Ad-375-infected islets. Finally, depoalrization with highextracellular K⁺ increased [Ca²⁺]_(i) to the same extent in control andAd-375-infected islets and the peak [Ca²⁺]_(i) averaged 0.85±0.24 μM and1.01±0.31 μM, respectively (data not shown). Qualitatively similarobservations were obtained using the non-ratiometric dye fluo-3 (seeabove). Moreover, no differences in glucose responsiveness were likewiseobserved when the measurements were carried out on eGFP-expressingisolated cells, excluding contribution of deeper non-infected celllayers (data not shown). The islet periphery is enriched in non-β-cells.However, the fact that no oscillations were observed at 5 mM glucose andthat an elevation to 15 mM increased [Ca²⁺]_(i) suggests that we hadselected a β-cell-rich zone as δ-cells would be active already at thelower concentration and α-cells should be inhibited at the higherconcentration.

The characterization of regulated insulin secretion from MIN6 cells andisolated islets indicated that miR-375 expression might affect insulinsecretion downstream of [Ca⁺⁺]i signaling, possibly at the level ofexocytosis. To address this hypothesis, we applied capacitancemeasurements to functionally-identified β-cells. Exocytosis was elicitedby a train of ten 500 ms depolarizations from −70 mV to 0 mV applied at1 Hz (FIG. 3A). In the β-cells, the train elicited an increase inmembrane capacitance of 837±244 fF (n=9) under control conditions. Incells infected with Ad-375, the corresponding increase was limited to94±27 fF (n=10; P<0.01), a decrease by 85%. Similar results was alsoobtained once exocytosis instead was induced by a Ca²⁺/EGTA-buffer witha free Ca²⁺-concentration of 1.5 μM (FIG. 2D-E) and in these experimentsthe rate of capacitance increase was reduced by 63% (P<0.001; n=15-17)in Ad-375-infected cells compared to the control cells.

Example 5 Identification of Target Genes

We applied an algorithm which combines thermodynamics-based modeling ofRNA:RNA duplex interactions with comparative sequence analysis topredict microRNA targets conserved across multiple genome. From thecompiled list of 64 putative miR-375 target genes, we selected six genesbased on their potential role in insulin secretion/islet differentiationfor validation studies.

These genes included the frizzled homolog-4 (Fzd4), vesicle transportthrough interaction with t-SNAREs yeast homolog 1A (Vti-1a),V-1/myotrophin (V-1/Mtpn), p38 mitogen-activated protein kinase(Mapk11), monocarboxylic acid transporter member 8 (Slc16A2) and thepaired box protein Pax-6. The expression of these genes was studied byimmunoblotting in MIN6 and N2A cells that were infected with eitherAd-375 or Ad-eGFP. Expression of miR-375 in N2A cells, which do notexpress endogenous miR-375, led to a reduction in expression levels ofMtpn and Vti-1a (FIG. 4A, B).

In contrast, gene expression of Fzd4, Mapk11 and Slc16A2 were equivalentin Ad-375 and Ad-eGFP infected cells. Overexpression of miR-375 in MIN6cells using recombinant adenovirus Ad-375 also decreased protein levelsof Mtpn but had no effect on the expression of Vti-1a, Fzd4, Mapk11 andSlc16A2.

To investigate if the predicted miR-375 target site in the 3′ UTR of theMtpn mRNA was responsible for silencing of Mtpn expression by miR-375,we cloned a 289 nt 3′UTR segment that included the putative 3′ UTRtarget site downstream of a Renilla luciferase ORF (pRL-Mtpn) andco-transfected this reporter vector into MIN6 cells with a controlantisense 2′-O-methyl oligoribonucleotides or an miR-375 antisense2′-O-methyl oligoribonucleotide (2′-O-miR-375). Luciferase activity ofcells transfected with the 2′-O-miR-375 was ˜2-fold increased comparedto cells that were co-transfected with control 2′-O-miRNA and pRL-Mtpn.Furthermore, creating point mutations in the core of the miR-375 targetsite, thereby reducing the complementarity between endogenous miR-375and the V-1/myotrophin target site, abolished the stimulatory action of2′-O-miR-375 on luciferase activity (FIG. 5). Therefore, Mtpn is atarget of miR-375 in pancreatic β-cells and the repression of Mtpn geneexpression is mediated by a single miR-375 target site in the 3′ UTR ofthe Mtpn gene.

To test if decreased expression of Mtpn in MIN6 cells may contribute tothe defect in glucose-induced insulin secretion observed in Ad-375infected cells, MIN6 cells were transfected with siRNAs targetingapolipoprotein M (control) and Mtpn and protein expression levels wereassayed by western blotting. Both siRNAs targeting Mtpn and Vti1areduced expression of these genes by >50% (FIG. 4 c). The effect of genesilencing of these genes on glucose-induced insulin secretion wasstudied by co-transfection of the respective siRNA and plasmid pCMV-hGH.Insulin secretion in response to a 25 mM glucose challenge was measured2 days after transfection. Insulin secretion was reduced ≈30% in cellstransfected with siRNA targeting Mtpn compared to cells that weretransfected with control siRNA (FIG. 4 d). RNA silencing of Vti1a had noeffect on glucose-induced insulin secretion.

INCORPORATION OF SEQUENCE LISTING

Incorporated herein by reference in its entirety is the Sequence Listingfor the application. The Sequence Listing is disclosed on acomputer-readable ASCII text file titled, “sequence_listing”, created onMar. 10, 2014. The sequence_listing.txt file is 13.2 kb in size.

What is claimed is:
 1. An isolated nucleic acid molecule consisting ofan anti-microRNA that is 100% complementary to SEQ. ID. NO. 10, whereinthe molecule comprises at least one modified moiety for increasednuclease resistance.
 2. A molecule according to claim 1, wherein atleast one of the moieties is a modified ribonucleotide moiety.
 3. Amolecule according to claim 2, wherein the modified ribonucleotide issubstituted at the 2′ position.
 4. A molecule according to claim 3,wherein the substituent at the 2′ position is a C₁ to C₄ alkyl group. 5.A molecule according to claim 4, wherein the alkyl group is methyl.
 6. Amolecule according to claim 4, wherein the alkyl group is allyl.
 7. Amolecule according to claim 3, wherein the substituent at the 2′position is a C₁ to C₄ alkoxy-C₁ to C₄ alkyl group.
 8. A moleculeaccording to claim 7, wherein the C₁ to C₄ alkoxy-C₁ to C₄ alkyl groupis methoxyethyl.
 9. A molecule according to claim 2, wherein themodified ribonucleotide has a methylene bridge between the 2′-oxygenatom and the 4′-carbon atom.
 10. A molecule according to claim 1,wherein at least one of the moieties is a peptide nucleic acid moiety.11. A molecule according to claim 1, wherein at least one of themoieties is a 2′-fluororibonucleotide moiety.
 12. A molecule accordingto claim 1, wherein at least one of the moieties is a morpholinophosphoroamidate nucleotide moiety.
 13. A molecule according to claim 1,wherein at least one of the moieties is a tricyclo nucleotide moiety.14. A molecule according to claim 1, wherein at least one of themoieties is a cyclohexene nucleotide moiety.
 15. A molecule according toclaim 1, wherein the nuclease is an exonuclease.
 16. A moleculeaccording to claim 15, wherein the molecule comprises at least onemodified moiety at the 5′ end.
 17. A molecule according to claim 15,wherein the molecule comprises at least two modified moieties at the 5′end.
 18. A molecule according to claim 15, wherein the moleculecomprises at least one modified moiety at the 3′ end.
 19. A moleculeaccording to claim 15, wherein the molecule comprises at least twomodified moieties at the 3′ end.
 20. A molecule according to claim 15,wherein the molecule comprises at least one modified moiety at the 5′end and at least one modified moiety at the 3′end.
 21. A moleculeaccording to claim 15, wherein the molecule comprises at least twomodified moieties at the 5′ end and at least two modified moieties atthe 3′end.
 22. A molecule according to claim 1, wherein the nuclease isan endonuclease.
 23. A molecule according to claim 22, wherein themolecule comprises at least one modified moiety between the 5′ and 3′end.
 24. An isolated nucleic acid molecule consisting of the sequence ofbases of SEQ ID NO: 10, wherein the molecule comprises at least onemodified moiety for increased nuclease resistance.
 25. A vectorcomprising the molecule of claim
 1. 26. A vector comprising the moleculeof claim 24.